BackgroundThe presence of autoantibodies to angiotensin 2 type 1 receptor (anti-AT1R) have been implicated in allograft pathobiology following organ transplantation. While the significance of these antibodies has been described in renal transplantation, relatively few studies have examined their frequency and clinical implications in heart transplant patients.MethodsWe analyzed serum collected from 291 heart transplant recipients at the time of transplantation for the presence of anti-AT1R and repeated testing on serum collected from 176 of these patients following transplantation. Patients were followed for outcomes including overall survival, rejection episodes (acute cellular and antibody mediated), coronary allograft vasculopathy, and measures of allograft structure and cardiac function.ResultsAnti-AT1R was detected in the serum of 165/291 patients pre-transplant and in 86/176 patients post-transplant. The detection of anti-AT1R (either at risk or positive) compared with no detection in serum of patients pre- or post-transplantation had no influence on 10-year survival (Log rank 0.061 and 0.228, detection pre- or post-transplant, respectively). Similarly, the detection of anti-AT1R had no influence on important clinical outcomes of heart transplantation including acute cellular rejection (ACR), antibody-mediated rejection (AMR) or cardiac allograft vasculopathy (CAV), left ventricular ejection fraction (LVEF) or left ventricular mass (LV-mass).ConclusionsThe presence of anti-AT1R detected in patient serum samples by commercially available testing pre- or post- heart transplantation was not associated with clinically important outcomes including LV-mass, LVEF, ACR, AMR, CAV and overall survival. Our data brings into question the relevance of anti-AT1R testing as a risk factor or target for therapy among heart transplant recipients.

Abstract Aims Fibroblast to myofibroblast trans‐differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de‐differentiation could mitigate CF‐related pathologies. We determined whether 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase inhibitors—statins, commonly prescribed to patients at risk of heart failure (HF)—can de‐differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients. Methods and results Either in vitro statin treatment of differentiated myofibroblasts (n = 3–6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4–12). In vitro, hVFs were differentiated by transforming growth factor‐β1 (TGF‐β1) for 72 h (TGF‐72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α‐smooth muscle actin (α‐SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P < 0.05, considered significant. In vitro, statins concentration‐dependently de‐differentiated the myofibroblasts. The respective half‐maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 μmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 μmol/L), the reduced product of HMG‐CoA, prevented the statin‐induced de‐differentiation (α‐SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 μmol/L), a cholesterol synthesis‐independent HMG‐CoA reductase pathway intermediate, completely prevented the statin‐induced de‐differentiation (α‐SMA/GAPDH ratios: 0.89 ± 0.05 [TGF‐72 h + 72 h], 0.63 ± 0.02 [TGF‐72 h + simvastatin], and 1.2 ± 0.08 [TGF‐72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co‐incubation of simvastatin (200 nmol/L) with glibenclamide (10 μmol/L), a KATP channel inhibitor, attenuated the simvastatin‐induced de‐differentiation (0.84 ± 0.05). Direct inhibition of mitochondrial respiration by oligomycin (1 ng/mL) also produced a de‐differentiation effect (0.33 ± 0.02). OCR (pmol O2/min/μg protein) was significantly decreased in the simvastatin‐treated hVFs, including basal (P = 0.002), ATP‐linked (P = 0.01), proton leak‐linked (P = 0.01), and maximal (P < 0.001). The OCR inhibition was prevented by GGPP (basal OCR [P = 0.02], spare capacity OCR [P = 0.008], and maximal OCR [P = 0.003]). Congruently, hVFs from HF showed an increased population of myofibroblasts while HF + statin group showed significantly reduced cellular respiration (basal OCR [P = 0.021], ATP‐linked OCR [P = 0.047], maximal OCR [P = 0.02], and spare capacity OCR [P = 0.025]) and myofibroblast differentiation (α‐SMA/GAPDH: 1 ± 0.19 vs. 0.23 ± 0.06, P = 0.01). Conclusions This study demonstrates the de‐differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of KATP channels, and their impact on the differentiation magnitude of hVFs in HF patients. This novel pleiotropic effect of statins may be exploited to reduce excessive CF in patients at risk of HF.

Excessive fibrosis underlies many critical organ dysfunctions.1, 2 Fibrosis emanates from fibroblast trans-differentiation into myofibroblasts,3 marked by increased α-smooth muscle actin (α-SMA) expression and excessive collagen secretion, initiated as a reparative process of normal wound healing and tissue repair in response to injury.4 However, activated myofibroblasts accumulate within pathological lesions of various fibrotic disorders,5 including patchy and interstitial fibrosis in progressive heart failure and cardiac hypertrophy.6 Therefore, attenuation of differentiation to myofibroblasts is expected to mitigate fibrosis. We attempted to find a potential target to extenuate the fibroblast differentiation by analysing the transcription factors in human fibroblasts/myofibroblasts, as transcriptome changes occur in fibroblasts during differentiation.7 Here, we report a novel molecular target, transcription factor AP-2α (TFAP2A), to reduce fibroblasts trans-differentiation. Informed consents were obtained from all participants, and the study was carried out according to the World Medical Association Declaration of Helsinki. Human ventricular fibroblasts (hVFs)-Control hVFs from disease-free trauma victims (Lonza Inc, Allendale, NJ; ScienCell, Carlsbad, CA); hVFs were isolated from Heart Patients (Aurora Health Care, Milwaukee, WI), (HF) as reported earlier.8 NIH/3T3 fibroblasts (ATCC, Manassas, VA), Transforming growth factor (TGF)-β1 (Peprotech, Rocky Hill, NJ), angiotensin II (Abcam, Cambridge, MA), miRNeasy Mini Kit, RT2 Profiler PCR Array, RT2 First Strand Kit, RT2 SYBR Green PCR master mix, miScript II RT kit (QIAGEN, Venlo, the Netherlands); Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA), Antibodies: Anti-α-SMA, Anti-TFAP2A (Abcam, Cambridge, MA), Anti-α/β-tubulin and Anti-GAPDH (Cell Signaling, Danvers, MA) were purchased. The isolated hVFs were grouped into fibroblasts-less differentiated (HF-LD) and fibroblasts-highly differentiated (HF-HD) based on their α-SMA expression (immunoblot), compared to the control hVFs (Figure 1A,B). Polymerase chain reaction (PCR) array was performed with RT2 Profiler™ PCR Array-Human Transcription Factors and compared between HF-LD (n = 3) and HF-HD (n = 3). Mature RNA (miRNeasy Mini kit) was reverse transcribed using RT2 First strand cDNA synthesis kit. The cDNA was used on the real-time RT2 Profiler PCR Array (QIAGEN, Cat# PAHS-075Z) in combination with RT2 SYBR® Green qPCR Mastermix (Roche LightCycler® 480 Instrument). Threshold cycle (CT) values (excel file) were uploaded onto the data analysis centre web portal (http://www.qiagen.com/geneglobe). CT values were normalized based on a Manual Selection of reference genes. The fold change/regulation (2^(-ΔΔCT)) was calculated using ΔΔCT method [ΔCT was calculated between target gene and an average of reference genes (HKG), followed by ΔΔCT calculations (ΔCT (Test Group)-ΔCT (Control Group))]. Total RNA was isolated from hVFs (miRNeasy Mini kit) and reverse transcribed (miScript RT II kit) with the supplied HiFlex buffer. qPCR was performed on the LightCycler 480 Instrument II, using the Power SYBR Green PCR Master Mix and 10 ng diluted cDNA per well. The following human primers were used: TFAP2A—F:5′-GACCTCTCGATCCACTCCTTAC-3′ R: 5′-GAGACGGCATTGCTGTTGGACT-3′; β-2-microglobulin (B2M)- F: 5′-CCACTGAAAAAGATGAGTATGCCT-3′ and R: 5′-CCAATCCAAATGCGGCATCTTCA-3′. The following PrimeTime qPCR mouse primer assays were used: α-SMA (Mm.PT.58.16320644); COL1A1 (Mm.PT.58.7562513); COL2A1 (Mm.PT.58.5206680); COL3A1 (Mm.PT.58.13848686), TGFBR1 (Mm.PT.58.28402453), TGFBR2 (Mm.PT.58.6358355) and B2M (Mm.PT.39a.22214835). The cycling conditions were 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 s, 1 minute at 60°C, and 72°C for 40 s. Melt curve analysis was performed by an additional dissociation step of 1 cycle at 95°C for 5 s followed by 65°C for 1 min and ramping data collection to 97°C. Relative expression values (ΔCt) were obtained by normalizing Ct values (Roche Lightcycler 480 Software v1.5.1.62) of the tested genes with that of B2M. The TFAP2A knockout cell line with NIH/3T3 fibroblasts (TFAP2A-KO) was established using CRISPR/CAS9 technology through Creative Biogene, Shirley, NY. Fibroblasts from wild-type or TFAP2A-KO groups were plated at 4000 cells/cm2 with DMEM media (10% BCS) and incubated at 37°C under 5%CO2. Following 24 hours, hVFs were either treated with TGF-β1 (5 ng/mL), angiotensin II (100 nM) or kept as control in DMEM media (2.5% BCS). After 48-72 hours, the fibroblasts/myofibroblasts were rinsed with Dulbecco's PBS and assayed. Standard western protocols were followed8 with respective primary (dilutions: α-SMA, 1:500, TFAP2A, 1:100) and secondary antibodies (1:2000). All samples were immunoblotted simultaneously and repeated at least twice. Both WT and KO fibroblasts were plated as stated before in triplicate (per time-point) in 6-well plates and counted by Cellometer Auto 2000 (Nexcelom Bioscience, Lawrence, MA) at 24, 48, and 72 hours post-plating. Doubling time was calculated by [t − t0]/{[log(Nt)-log(N0)]/log(2)}, where t0 refers time (initial count), t represents time (second count), N0 refers count at time t0, and Nt represents count at time t. From left ventricle of human heart, fibroblasts were isolated and grouped into less differentiated (HF-LD) and highly differentiated (HF-HD) based on their α-SMA expression, compared to control hVFs as shown in Figure 1A,B. PCR array of human transcription factors uncovered that the TFAP2A expression, along with ELK1, was decreased with decrease in differentiation as visualized in the heat map (Figure 1C) and fold regulation data (Figure 1D) (n = 3). This decreased expression of TFAP2A in HF-LD fibroblasts compared to HF-HD myofibroblasts noticed in PCR array was validated by quantitative reverse transcriptase-PCR (n = 5) (Figure 1E). Based on these data, we have suggested that TFAP2A is crucial for the trans-differentiation of fibroblasts into myofibroblasts. We applied CRISPR/Cas9-based gene editing to knockout TFAP2A from NIH/3T3 fibroblasts (Figure 2A) and analysed the differentiation- and pro-fibrotic parameters at both basal level and following TGF-β1 treatment. TGF-β1 significantly increased the mRNA expression of α-SMA (Figure 2B), collagen (COL) 1A1 (Figure 2C), COL2A1 (Figure 2D) in the wild-type while the TGF-β1 effect was significantly low in the TFAP2A-KO fibroblasts. Even at basal level, the expressions of α-SMA (Figure 2B) and COL3A1 (Figure 2E) were significantly decreased in the TFAP2A-KO fibroblasts compared to the wild-type. This suggests that TFAP2A is important for the trans-differentiation of fibroblasts to myofibroblasts. This reduced differentiation of TFAP2A-KO fibroblasts observed in qPCR was further confirmed at protein level by immunoblotting where α-SMA expression was significantly low both at basal level and after TGF-β1 administration (Figure 2F,G). The blunted effect of TGF-β1 in the TFAP2A-KO fibroblasts does not appear to be due to changes in the upstream TGF-β1 receptor levels, as the mRNA levels of TGF-β1 receptor type1 (TGFBR1) is increased in TFAP2A-KO fibroblasts (Figure 2H) without any significant difference in the type2 receptors (TBFBR2) compared to the wild-type (Figure 2I). Interestingly, deletion of TFAP2A gene attenuates not only TGF-β1-induced fibroblast differentiation, but also angiotensin II (Ang II)-induced differentiation as well, as evident from lack of increase in α-SMA expression in the TFAP2A-KO fibroblasts (Supplemental Figure). This suggests that TFAP2A could serve as a common downstream regulator of genes associated with fibroblast differentiation. Importantly, the knockdown of TFAP2A did not adversely affect the basal proliferation capacity (Figure 2J). The TFAP2A-KO fibroblasts proliferated like that of the wild-type with a doubling time of 21 ± 6 hr (TFAP2A-KO) vs 25 ± 7 hr (wild-type) (n = 3). TFAP2A is a known DNA-binding transcription factor to have both repressive and facilitating effects9 on various genes and complete knockout of which is embryonically lethal.10 The exact mechanism for the reduced trans-differentiation of TFAP2A-KO fibroblasts in response to TGF-β1 is unclear. Chromatin immunoprecipitation studies of Smad2/3, important factors in TGF-β1 signalling, revealed abundant TFAP2A binding elements in Smad2/3 binding sites of the promoter regions of various genes in keratinocytes and knockdown of TFAP2A changed the TGF-β1- induced transcriptions.11 Whether similar mechanisms underlie in fibroblasts is not known. In human Sertoli cells, Bone Morphogenetic Protein (BMP) 6, a member of TGF-β superfamily, targets TFAP2A to positively regulate their growth.12 In contrast, the basal proliferation of fibroblasts did not reduce following TFAP2A knockdown in our study. This is in accordance with the observation in another study where TFAP2A can induce cell cycle arrest13 while reduced TFAP2A expression was suggested to impair p21cip-mediated growth arrest resulting in increased proliferation.14 These properties found in the TFAP2A-KO fibroblasts suggest that TFAP2A could emerge as a useful molecular target to mitigate excessive fibrosis by inhibiting fibroblast differentiation. As evident from the isolated human cardiac fibroblasts from left ventricles, the decrease in TFAP2A expression when cardiac fibroblast differentiation is decreased, suggest that TFAP2A is crucial for the trans-differentiation of cardiac fibroblasts into myofibroblasts which can lead to excessive cardiac fibrosis underlying many cardiac dysfunctions. Therefore, selective inhibition of TFAP2A could develop as a novel therapeutic strategy to reduce cardiac fibroblast differentiation into myofibroblast, mitigate cardiac fibrosis and preserve cardiac function. Aurora Health Care Cardiovascular Surgery Research Award (#570-5028) to GRR. There is no conflict of interest. GRR initiated, designed, executed, analysed the study and wrote the manuscript; SE executed the real-time PCR and PCR array; CW and PH implemented the cell culture, immunoblotting and proliferation assays; FXD, LE, FR and AJ interpreted data and proof-read the manuscript. All data sets are publicly available from the Dryad Digital Repository at https://doi.org/10.6084/m9.figshare.7898168. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: The MOMENTUM 3 study (Multicenter Study of MagLev Technology in Patients Undergoing Mechanical Circulatory Support Therapy With HeartMate 3) has demonstrated that the HeartMate 3 (HM3) pump is associated with reduced strokes compared with the HeartMate II (HMII) device. We now perform a comprehensive analysis of stroke events to evaluate their longitudinal occurrence, clinical correlates, patterns, and impact on outcome across the 2-year duration of support. Methods: MOMENTUM 3 is a randomized controlled trial of the HM3 centrifugal-flow pump versus the HMII axial-flow pump in patients with advanced heart failure, regardless of the intended goal of support (bridge to transplantation or destination therapy). Baseline and postimplantation clinical correlates of stroke events were assessed with multivariable analyses. Longitudinal patterns, including device association, type of stroke (hemorrhagic versus ischemic), changing severity of impairment assessed with the modified Rankin Scale (disabling [modified Rankin Scale score >3] versus nondisabling [modified Rankin Scale score ≤3]) over time, and association with outcome, were determined. Results: In 361 patients with the intended implant (189 HM3 and 172 HMII), 65 strokes (40 ischemic strokes and 25 hemorrhagic strokes) occurred in 52 patients at a median of 131 (range, 1–733) days. No difference in stroke rate was noted between 0 and 180 days of follow-up between devices. However, stroke incidence in the long-term period (181–730 days after left ventricular assist device) was 3.3 times lower for the HM3 group (HM3: 0.04 versus HMII: 0.13 events per patient-year; odds ratio, 0.23; 95% CI, 0.08–0.63; P =0.01). Treatment with the HM3 pump was the only independent predictor of lower stroke events. We found no direct association of blood pressure or antithrombotic regimens with observed stroke rates. A stroke event significantly lowered 2-year postimplantation survival regardless of subtype or initial severity of neurological impairment compared with patients without a stroke (43±12% for hemorrhagic stroke, 57±9% for ischemic stroke, 51±11% for disabling, and 51±11% for nondisabling compared with 85±2% 2-year survival for patients without stroke). Conclusions: The HM3 pump is associated with a marked reduction in stroke rates compared with the HMII device, with benefits observed in the long-term period (>6 months). The occurrence of stroke of any type (hemorrhagic and ischemic) or of any functional severity (disabling and nondisabling) is predictive of a poor 2-year clinical outcome. Clinical Trial Registration: URL: https://www.clinicaltrials.gov/ . Unique identifier: NCT02224755.

Background: Circulating microRNAs have recently been identified as biomarkers predicting A-Fib and heart failure. Yet, their role in identifying those at risk for postoperative atrial fibrillation (PoAF) in a gender-specific manner has not been studied. Methods: Preoperative blood from patients undergoing cardiac surgery with no prior history of AF, supraventricular/ventricular tachycardia was used for RNA isolation. Differences in the relative levels of miRNA between those who did/did not develop PoAF were assessed using qPCR. Principal component analysis and receiver operative characteristics were performed to predict miRNA associated with PoAF. Results: Out of 56 patients, 29 developed PoAF (52%, mean age 70.4± 9y, 51% males), while 27 (mean age 63y ± 10) remained free of AF. Patients who developed PoAF were older (P<0.01). There were no differences in the prevalence of hypertension, diabetes, dyslipidemia, prior myocardial infarction, heart failure, stroke, COPD, or cardiac medications. Out of 13 miRNAs analyzed, differential levels of miR423 (AUC83.7%, CL 72.4% - 93.3%), miR187 (AUC 76.8%, CL 63.4% - 90.2%) and miR34a2 (AUC 67.7%, CL53%-82.4%) were associated with PoAF. Gender-specific analysis showed a different set of circulating miRNAs (miR-187,-34a2,-25) in male vs. female (miR-29a1, -21,-126,-15b) patients with miR423 common in both as an independent predictor of PoAF. Conclusion: This indicates the existence of different substrate/precipitating factors reflected in miRNAs profile in male and female patients who developed PoAF. Gender differences exist in circulatory miRNA levels can help in predicting the development of new-onset AF after cardiac surgery.

Autoantibodies to the angiotensin II type 1 receptor (AT1R) are thought to be important in antibody-mediated rejection (AMR), especially in the absence of anti-HLA antibodies. We used a variety of methods to examine the specificity of a commercially available kit designed to quantitate anti-AT1R antibodies. We found that fibrin formation in serum samples from patients awaiting cardiac transplantation with ventricular assist devices (VADs) can produce falsely elevated anti-AT1R values. In addition, absorption studies with a variety of cell lines with or without expression of human AT1R, and those that express xenoantigens, suggest that many of the antibodies detected in the AT1R test system are heterophilic and have reactivity to xenoantigens. Furthermore, we provide data that show that reactivity to the sialic acid Neu5Gc is a common finding among samples that are highest in anti-AT1R levels. We conclude that a common laboratory method for quantitation of anti-AT1R antibodies is nonspecific and overestimates the frequency of true positives. A reevaluation of the role that anti-AT1R antibodies play in allograft function and patient outcomes is warranted.

The association between sleep disordered breathing (SDB) and ventricular arrhythmias (VA) is well established. Limited data is available on the severity of SDB and VA among patients with continuous flow left ventricular assist device (CF-LVAD). Among 230 patients who received CF-LVAD between Jan

HeartWare HVAD log files provide a record of important diagnostics that assist in patient care. We believe that information regarding battery function can inform providers of patient behavior, compliance and future adverse events.

Arrhythmias resulting in internal cardiac defibrillator (ICD) shock are unpredictable, common (15-30%) and lead to increased morbidity and mortality among LVAD supported patients. We sought to evaluate the incidence and temporal implications of appropriate ICD discharge among patients supported with CF-LVAD.

Introduction: Right and left atria have different susceptibilities toward developing atrial fibrillation (AF). The molecular bases of these differences are not well characterized. Given the complexity of AF development and progression, understanding AF-associated changes in myocardial energetics between the atria will help improve mechanistic insights and therapeutics for better clinical management of AF. The aim was to compare changes in mitochondrial oxidative phosphorylation system (OXPHOS), glycolysis and Krebs cycle metabolites in right atrial (RAA) and left atrial (LAA) appendage tissue from patients with (AF) and without (non-AF) AF. Methods: RAA and LAA from well-matched AF (n=54) and non-AF (n=58) patients undergoing elective open heart surgery was collected. Functional activity of OXPHOS complexes I-V was measured spectrophotometrically. Protein expression level of OXPHOS complexes was determined by Western blot. OXPHOS gene expression level was performed using RT-PCR. Metabolites were profiled using high performance liquid chromatography coupled to tandem mass spectrometry. Comparison between groups was done applying the 2 sample t - and Wilcoxon rank sum tests with 5% level of significance. Results: The most significant AF-associated alterations in myocardial energetics were observed in RAA. In AF patients, out of 84 OXPHOS genes, expression of 14 genes was significantly reduced in RAA (p<0.05) and 2 genes was reduced in LAA (p<0.05). There was AF-associated reduction in complex I (p=0.01) and IV (p=0.04) protein expression in RAA without changes in LAA. Unlike LAA, mitochondria from RAA revealed decline in complex I (p=0.01) and II (p=0.03) activity in AF compared to non-AF patients. In AF patients, glycolysis metabolites level of glucose-6-phosphate and phosphoenolpyruvate was reduced in RAA (p=0.03), whereas 2-phosphoglycerate was reduced in LAA (p=0.02). AF was related with decrease in NAD + (p=0.03), GDP (p=0.05), citrate (p=0.03), total pool of adenine nucleotides (p=0.02) and glutathione (p=0.03) level in RAA without changes in LAA. Conclusion: AF is associated with different energetic remodeling in right and left atria, suggesting that dissimilar mechanisms may contribute to development and progression of AF.

Background: Several studies have demonstrated genomic, morphological, and electrophysiological differences between the right atrium and left atrium, suggesting that dissimilar mechanisms may contribute to the development and progression of atrial fibrillation (AF). Therefore, differences in metabolic response to AF between atria are foreseeable. Given the complexity of AF development and progression, understanding AF-associated changes in metabolites in both atria will help in better clinical management of AF.

Gastrointestinal bleeding (GIB) after continuous-flow left ventricular assist devices (CF-LVAD) is common (20-40%) and leads to increased morbidity and mortality. Previously we identified chronic obstructive pulmonary disease (COPD) as an independent risk factor for the development of GIB (HR=1.89, 1.10-3.23 P<0.02). This study explores pulmonary function test (PFT) parameters associated with GIB.

Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for [Ca2+]i in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF vs normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 hours of culture compared with control [85.9±7.4 μg/106 vs 58.5±8.8 μg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; rt-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry [SOCE /CRAC (Ca2+ release-activated)] channels was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF vs control hVFs was eliminated by incubation of hVFs with YM58483 (10 μM), a selective ICRAC inhibitor for 48 hours (66.78±5.87μg/106 cells vs 55.81±7.09 μg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity vs non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.

CF-LVAD improve quality and quantity of life for most patients. However, gastrointestinal bleeding (GIB) occurs in up to 30% of patients. Determine whether GI evaluation and specific pathologic lesions predict 12 month incidence of GIB. Retrospective single center study (1/06-12/14) found 254 patients who survived at least 15 days after CF-LVAD implant. Primary endpoint was 12 month incident GIB sensored for LVAD exchange, heart transplant or death. Descriptive statistics and cox proportional hazard were used to create univariate and multivariate models. A total of 63 (25%) patients had GIB after CF-LVAD. A cohort of 126 had GI evaluation prior to CF-LVAD. Univariant predictors of GIB with HR included: Age (1.45), CKD (1.78), HTN (2.12), prior CABG (2.14), prior PCI (2.74), prior GI abnl (2.05). Multivariable Cox predictors of GIB were prior GI abnl (2.19) and prior PCI (2.71). Among the 128 patients who underwent GI evaluation multivariable cox predictors of GIB were prior PCI (5.07 and prior GI abnl (2.53) persisted. In a large single center cohort we describe history of GI abnormality and prior PCI are statistically associated with 12 month incidence of GIB. We advocate closer examination of these factors as potential targets of therapy.

Introduction: Dronedarone (DR), a structural analog of amiodarone designed to reduce its extracardiac toxicity, recently was shown in clinical trials to increase heart failure (HF) and cardiovascular death in patients with severe myocardial dysfunction, but the mechanisms for these adverse cardiac effects are not known. Hypothesis: We hypothesized that DR increases oxidative stress and cell death in failing hearts with compromised energetic reserves compared to nonfailing hearts. Methods: Ventricular fibroblasts (VFB) from patients without HF (trauma victims) and with HF (left ventricular assist device implants) were cultured under similar conditions to passage 3 and treated with DR (0-10 μM) for 24h. Reactive oxygen species (ROS) production was assessed in cells loaded with general oxidative stress indicator CM-H2DCFDA (2μM), mitochondrial superoxide indicator MitoSOX Red (5 μM), and Hoechst 33342 (1μg/ml) for nuclear staining using confocal microscopy. Cell viability was assessed by counting the number of viable cells and lactate dehydrogenase assay. Results: VFB from HF patients exhibited a higher baseline level of total ROS (CM-H2DCFDA green fluorescence) when compared with non-HF patients (Fig. A). DR dose-dependently increased ROS production in VFB from HF patients (Fig. B) exhibiting a higher sensitivity to oxidative stress and cell death (Fig. C). Conclusions: The sensitivity of DR-induced oxidative stress and cytotoxicity is significantly increased in VFB from patients with HF and provides an explanation for adverse effects of DR reported in HF patients in clinical trials.

Introduction: Continuous-flow Left Ventricular Assisted Device (CF-LVAD) therapy is increasingly utilized for patients with end stage heart failure. Among the most common and unpredictable complications after CF-LVAD is gastrointestinal bleeding (GIB). Hypothesis: We hypothesize that pre-implant characteristics are associated with GIB post-implant of a CF-LVAD. The aim of this analysis is to identify novel pre-implant factors that influence risk of post-implant GIB. Methods: All CF-LVAD implants between January 2006 and December 2014 among patients who survived more than 15 days were included and followed for 12 months. Primary event was GIB and patients were censored at time of re-implant, heart transplant, or death. Student’s t-test was used to compare continuous variables and chi-square test for categorical variables. Cox Proportional Hazards model was used to identify univariate and multivariable models predicting GIB. Results: Among the total 257 patients included, 65 (25.3%) were identified as having a GIB. Baseline differences and their independent univariate hazard ratio (HR) are noted in Table 1. Using stepwise selection and developing a multivariable model, prior GI abnormalities (HR=2.12, p<0.01), prior percutaneous coronary intervention (PCI: HR=2.65, P<0.01) and chronic obstructive pulmonary disease (COPD: HR=1.73, P<0.01) remained statistically significant predictors of GIB (Table 2). Conclusions: We describe novel risk factors (history of GI abnormalities, prior PCI, COPD) as predictors for developing GIB post CF-LVAD implantation. We advocate a closer examination of these risk factors when evaluating patients for CF-LVAD therapy.

Introduction: Left ventricular assist device (LVAD) is being increasingly utilized in patients with advanced heart failure both as bridge to heart transplantation and as destination therapy. Intracranial hemorrhage (ICH) is one of major complications associated with LVAD. However, current trends on utilization of LVAD and associated ICH in real world practice are not known. Methods: We analyzed patients in the Nationwide Inpatient Sample (NIS) between 2007 and 2011. Heart failure patients with LVAD were identified from the database and patients with discharge diagnosis of ICH were compared to those without ICH. Trends and outcomes of ICH in patients with LVAD were analyzed. In addition, predictors of ICH were identified using a multivariate regression model. Results: We identified 20,443 discharges with a primary diagnosis of heart failure with LVAD of which 447 patients had a co-diagnosis of ICH. We saw a significant increase in discharge diagnosis of heart failure with LVAD from 1232 discharges in 2007 to 6308 in 2011 (p<0.001)(Figure). However, the incidence of ICH in this patient population decreased from 3.9% in 2007 to 2.4% in 2011. On multivariate analysis, in-hospital mortality was significantly higher in the ICH group (OR: 9.5, P=0.0001). After adjustment for potential confounders age <35 years (OR: 2.4, P=0.01) and a rising Charlson co-morbidity index score were independent predictors of ICH whereas presence of diabetes (OR: 0.05, P=0.001), chronic lung disease (OR:0.07, P=0.001), renal disease (OR: 0.09, P=0.001) and peripheral vascular disease (OR: 0.12, P=0.002) were found to be protective of ICH. Conclusions: Our analysis indicates an increasing trend in utilization of LVAD but a decrease in incidence of ICH over the same period. ICH was found to increase risk of mortality by nearly 10 fold. Increasing comorbidity burden increases the risk of ICH whereas age and certain individual co-morbidities appear to have a paradoxical effect on risk of ICH.